Method of treating cancer and other proliferative diseases with a gold(I) complex

ABSTRACT

The invention is directed to a method of treatment of proliferative diseases or disorders such as cancer with gold(I) N-heterocyclic carbene (NHC) thiourea or substituted thiourea complexes, to the complexes per se and to therapeutic compositions containing these gold(I) based complexes.

STATEMENT OF FUNDING ACKNOWLEDGEMENT

This research was supported by the King Fahd University and Minerals(KFUPM) Research Committee under project No. IN171005.

REFERENCE TO A SEQUENCE LISTING

In accordance with 37 CFR § 1.52(e)(5), the present specification makesreference to a Sequence Listing which is submitted electronically as a.txt file named “528178US_ST25.txt”. The .txt file was generated on Feb.27, 2020 and is 1.42 kb in size. The entire contents of the SequenceListing are herein incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The invention falls within the field of medicine as directed totreatment of proliferative diseases or disorders such as cancer withgold(I) N-heterocyclic carbene (NHC) thiourea or substitute thioureacomplexes and to therapeutic compositions containing these gold(I) basedcomplexes.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

Metal-based pharmaceutical agents (Pd, Pt, Cu, Au, Ag) have beendocumented for their usefulness as antimicrobial and anticancer drugs.Among them, gold complexes have recently demonstrated significantbiological activities and have been used to develop novel therapeuticagents. See V. Gandin, F. Tisato, A. Dolmella, M. Pellei, C. Santini, M.Giorgetti, C. Marzano, M. Porchia, and C. S. Uniti, “In Vitro and inVivo Anticancer Activity of Copper(I) complexes with homoscorpionatetridentate tris(pyrazolyl)borate and auxiliary monodentate phosphineligands,” J. Med Chem., 2014, 12(57), 4745-60; P. Bippus, M. Skocic, M.A. Jakupec, B. K. Keppler, F. Mohr, “Synthesis, structures and in vitrocytotoxicity of some cationic cis-platinum(II) complexes containingchelating thiocarbamates,” J. Inorg. Biochem., 2011, 105(3), 462-466; W.Liu, K. Bensdorf, M. Proetto, U. Abram, A. Hagenbach, R. Gust, “NHC goldhalide complexes derived from 4,5-diarylimidazoles: synthesis,structural analysis, and pharmacological investigations as potentialantitumor agents,” J. Med. Chem., 2011, 54, 8605-8615; and J. Carlos, L.Rodriguez, “Phosphine-gold(I) compounds as anticancer agents: generaldescription and mechanisms of action,” Anti-Cancer. Agent. Med. Chem.,2011, 11, 921-928, each incorporated herein by reference in theirentirety. N-heterocyclic carbene (NHC) is a versatile derivative and itsmetal complexes have emerged as a focus of research for the developmentof catalysts and metallo-drugs due to their high stability. See C.Abbehausen, E. J. Peterson, R. E. De Paiva, P. P. Corbi, A. L. B.Formiga, Y. Qu, N. P. Farrell, “Gold(I)-phosphine-N-heterocycles:biological activity and specific (ligand) interactions on the C-terminalHIVNCp7 Zinc Finger,” Inorg Chem., 2013, 52, 11280-11287; H. D.Velazquez, F. Verpoort, “N-Heterocyclic carbene transition metalcomplexes for catalysis in aqueous media.,” Chem. Soc. Rev., 2012,41(21), 7032-60; E. Schuh, P. Carolin, A. Citta, A. Folda, M. P.Rigobello, A. Bindoli, A. Casini, F. Mohr, “Gold(I) carbene complexescausing thioredoxin 1 and thioredoxin 2 oxidation as potentialanticancer agents,” J. Med. Chem., 2012, 55, 5518-5528; X. Xu, S. H.Kim, X. Zhang, A. K. Das, H. Hirao, S. H. Hong, “Abnormal N-heterocycliccarbene gold(I) complexes: synthesis, structure, and catalysis inhydration of alkynes,” Organometallics, 2013, 32, 164-171; M. K.Samantaray, C. Dash, M. M. Shaikh, K. Pang, R. J. Butcher, P. Ghosh, N.York, U. States, “Gold (III) N-heterocyclic carbene complexes mediatedsynthesis of β-enaminones from 1,3-Dicarbonyl compounds and aliphaticamines,” Inorg. Chem., 2011, 50, 1840-1848; F. K. Keter, I. A. Guzei, J.Darkwa, “N-heterocyclic dithiocarbamate platinum(II) complexes:unexpected transformation of dithiocarbamate to oxodithiocarbonate inphosphinoplatinum complexes in solution,” Inorg. Chem. Commun., 2013,27, 60-63; D. G. Correia, F. E. Kiihn, B. Dominelli, “Medicinalapplications of gold(I/III)-based complexes bearing N-heterocycliccarbene and phosphine ligands,” J. Organomet. Chem., 2018, 866, 153-164;and C. Zhang, M. Maddelein, R. W. Sun, H. Gornitzka, O. Cuvillier, C.Hemmert, “European journal of medicinal chemistry pharmacomodulation ongold-NHC complexes for anticancer applications e is lipophilicity thekey point?,” Eur. J. Med Chem., 2018, 157, 320-332, each incorporatedherein by reference in their entirety.

L-ergothioneine is a thione-containing amino acid which naturally occursin the body. It performs important biological functions as anantioxidant and exists in two tautomeric forms. L-ergothioneine formscomplexes with several metal ions. See I. Erdelmeier, S. Daunay, R.Lebel, L. Farescour, J. Yadan, “Cysteine as a sustainable sulfur reagentfor the protecting-group-free synthesis of sulfur-containing aminoacids: biomimetic synthesis of L-ergothioneine in water” Green Chem.,2012, 14, 2256-2265; D. P. Hanlox, “Interaction of ergothioneine withmetal ions and metalloenzymes” J. Med. Chem., 1971, 14, 1084-1087; andT. N. Motohashi, I. Mori, Y. Sugiura, H. Tanaka, “Metal Complexes ofEgrothioneine,” Chem. Pharm. Bull., 1974, 22, 654-657, each incorporatedherein by reference in their entirety.

Mitochondria generate a large quantity of oxygen species (ROS) which areproduced in the cells and which are used for death signals originatingfrom intrinsic and extrinsic apoptosis. The biological activity of somegold compounds has been shown to be associated with mitochondriadysfunction, eventually leading to cancer cell death. For instance,reported gold(I)-NHC complexes possess different mechanisms of themitochondrial programmed cell death. See P. J. Barnard, M. V. Baker, S.J. Bemers-Price, D. A. Day, “Mitochondrial permeability transitioninduced by dinuclear gold(I)-carbene complexes: potential newantimitochondrial antitumour agents,” J. Inorg. Biochem., 2004, 98,1642-1647; A. Nandy, S. K. Dey, S. Das, R. N. Munda, J. Dinda, K. DasSaha, “Gold(I) N-heterocyclic carbene complex inhibits mouse melanomagrowth by p53 upregulation.,” Mol. Cancer, 2014, 13(1), 57; and H. G.Raubenheimer, S. Cronje, “Carbene complexes of gold: preparation,medical application and bonding,” Chem. Soc. Rev., 2008, 37(9),1998-2011, each incorporated herein by reference in their entirety.

An in vitro and in vivo study of thiourea coinage metal (Au, Ag, Cu)complexes was reported in which the Au(I) thiourea complex showed veryhigh potent tight-binding inhibition towards thioredoxin reductase. SeeC. Che, K. Yan, C. Lok, and C. Che, “Gold(I) complex ofN,N′-disubstituted cyclic thiourea with in vitro and in vivo anticancerproperties—potent tight-binding inhibition of thioredoxin reductase,”Chem. Commun., 2010, 46(41), 7691-7693, incorporated herein by referencein its entirety. The use of thiourea moiety in drugs as a tyrosinaseinhibitor by repositioning the thiourea-containing drugs through freenitrogen for intermolecular interaction was reported. See J. Choi, J.Jee, “Repositioning of thiourea-containing drugs as tyrosinaseinhibitors,” Int. J. Mol. Sci., 2015, 16, 28534-28548, incorporatedherein by reference in its entirety.

Recently, new studies of gold(I)-NHC complexes have reported showinganticancer activity. See O. Dada, G. Sanchez-sanz, M. Tacke, X. Zhu,“Synthesis and anticancer activity of novel NHC-gold(I)-sugarcomplexes,” Tetrahedron Lett., 2018, 59(30), 2904-2908; and A. Molter,S. Kathrein, B. Kircher, F. Mohr, “Anti-tumour active gold(T),palladium(II) and ruthenium(II) complexes with thio- and selenoureatoligands: a comparative study,” Dalt. Trans., 2018, 47, 5055-5064, eachincorporated herein by reference in their entirety. Altaf et al.synthesized a new class of gold(I)-NHC with dithiocarbamates complexesand evaluated their anticancer activity against A549, HCT15 and HeLacell lines. See M. Altaf, M. Monim-ul-mehboob, A. A. Seliman, A. A.Isab, V. Dhuna, G. Bhatia, and K. Dhuna, “Synthesis, X-ray structures,spectroscopic analysis and anticancer activity of novel gold(I) carbenecomplexes,” J. Organoniet. Chem., 2014, 765, 68-79, incorporated hereinby reference in its entirety. Moreover, Özdemir et al. synthesized aseries of gold(I)-NHC complexes. The complexes were evaluated for theirantimicrobial activity against Gram-positive, Gram-negative bacteria,and fungal species. See I. Özdemir, N. Temelli, S. Günal, S. Demir,“Gold(I) Complexes of N-heterocyclic carbene ligands containingbenzimidazole: synthesis and antimicrobial activity,” Molecules, 2010,15(4), 2203-2210, incorporated herein by reference in its entirety.

Extrinsic apoptosis is initiated through transmembrane death receptorsand the execution of these processes is mainly regulated by the BCL-2and caspase proteins. See K. S. Danial NN, “No Title,” Cell death Crit.Control points, vol. 116, no. 2, pp. 205-219, 2004; and L. Galluzzi, I.Vitale, J. M. Abrams, E. S. Alnemri, E. H. Baehrecke, M. V.Blagosklonny, T. M. Dawson, V. L. Dawson, W. S. El-Deiry, “Moleculardefinitions of cell death subroutines: recommendations of thenomenclature committee on cell death 2012,” Cell Death Differentiation,2012, 19(1), 107-120, each incorporated herein by reference in theirentirety. Activation of the BCL-2 family members results in the releaseof pro-apoptotic proteins, including cytochrome c, which ultimatelyactivates the caspase family proteins. See M. C. Wei, T. Lindsten, V. K.Mootha, S. Weiler, A. Gross, M. Ashiya, C. B. Thompson, “tBID, amembrane-targeted death ligand, oligomerizes BAK to release cytochromec,” Genes Dev., 2000, 14(16), 2060-71; M. C. Wei, W. X. Zong, E. H.Cheng, T. Lindsten, V. Panoutsakopoulou, A. J. Ross, K. A. Roth, G. R.MacGregor, C. B. Thompson, “Proapoptotic BAX and BAK: A requisitegateway to mitochondrial dysfunction and death,” Science 2001,292(5517), 727-730; and R. Eskes, S. Desagher, B. Antonsson, J-C.Martinou, “Bid induces the oligomerization and insertion of Bax into theouter mitochondrial membrane,” Mol. Cell Biol., 2000, 20(3), pp.929-935, each incorporated herein by reference in their entirety. Thenthese effector caspases ultimately lead to the hallmarks of apoptosis,including DNA fragmentation, cell shrinkage and membrane blebbing. SeeM. Woo, R. Hakem, M. S. Soengas, G. S. Duncan, A. Shahinian, D. Kagi, A.Hakem, M. McCurrach, W. Khoo, “Essential contribution of caspase 3/CPP32to apoptosis and its associated nuclear changes,” Genes Dev, 1998,12(6), 806-819, incorporated herein by reference in its entirety.

Gold compounds as inhibitors interact with thiol or selenol, the activesite of methionine and Se-methionine, which are used as antioxidants toprevent human cancers. See M. Bjornstedt, A. P. Fernandes, “Selenium inthe prevention of human cancers,” J. EPMA, 2010, 1(3), 389-95; H.Tapiero, D. Townsend, K. Tew, “The antioxidant role of selenium andseleno-compounds,” Biomed. Pharmacother., 2003, 57(3-4), 134-144; M.Navarro-Alarcon, C. Cabrera-Vique, “Selenium in food and the human body:a review.,” Sci. Total Environ., 2008, 400(1-3), 115-41; L. Letavayová,V. Vlcková, J. Brozmanová, “Selenium: from cancer prevention to DNAdamage.,” Toxicology, 2006, 227(1-2), 1-14; and R. Laplaza, V. Polo, J.Quero, S. Cabello, T. Fuertes, E. Cerrada, M. Concepcio, “Proteasomeversus thioredoxin reductase competition as possible biological targetsin antitumor mixed thiolate-dithiocarbamate gold(III) complexes,” Inorg.Chem., 2018, 57, 10832-10845, each incorporated herein by reference intheir entirety. Also, glutathione peroxidase (GPx), thioredoxin (Trx),and the enzyme thioredoxin reductase (TrxR) are used to protectdifferent organisms from damage due to the catalytic cycle reducingharmful peroxidase. See K. P. Bhabak, B. J. Bhuyan, G. Mugesh,“Bioinorganic and medicinal chemistry: aspects of gold(I)-proteincomplexes,” Dalt. Trans., 2011, 40, 2099-2111; S. E. Jackson-Rosario, W.T. Self, “Targeting selenium metabolism and selenoproteins: novelavenues for drug discovery.,” Metallomics, 2010, 2(2), 112-6; L.Oehninger, M. Stefanopoulou, H. Alborzinia, J. Schur, S. Ludewig, K.Namikawa, A. Muñoz-Castro, R. W. Köster, K. Baumann, S. Wölfl, W. S.Sheldrick, I. Ott, “Evaluation of arene ruthenium(II) N-heterocycliccarbene complexes as organometallics interacting with thiol and selenolcontaining biomolecules.,” Dalton Trans., 2013, 42(5), 1657-66; and J.L. Hickey, R. A. Ruhayel, P. J. Barnard, M. V Baker, S. J.Berners-price, A. Filipovska, “Mitochondria-Targeted Chemotherapeutics:The rational design of gold(I) N-heterocyclic carbene complexes that areselectively toxic to cancer cells and target protein selenols inpreference to thiols,” 2008, I, 12570-12571, each incorporated herein byreference in their entirety.

Previous studies evaluated the in vitro cytotoxicity against differentcancer cell lines and molecular docking with DNA of a series of gold(I)N-hetero-cyclic carbenes with aliphatic and hydrocyclic selenoneligands, which have higher anticancer activity than cisplatin; see A. A.A. Seliman, M. Altaf, N. A. Odewunmi, A. Kawde, W. Zierkiewicz, S.Ahmad, S. Altuwaijri, A. A. Isab, “Synthesis, X-ray structure, DFTcalculations and anticancer activity of a selenourea coordinatedgold(I)-carbene complex,” Polyhedron, 2017, 137, 197-206; A. A. A.Seliman, M. Altaf, A. T. Onawole, S. Ahmad, M. Yagoub, A. A. Al-saadi,S. Altuwaijri, G. Bhatia, J. Singh, A. A. Isab, “Synthesis, X-raystructures and anticancer activity of gold(I)-carbene complexes withselenones as co-ligands and their molecular docking studies withthioredoxin reductase,” J. Organomet. Chem., 2017, 848, 175-183; and A.A. A. Seliman, M. Altaf, A. T. Onawole, A. Al-saadi, S. Ahmad, A.Alhoshani, G. Bhatia, A. A. Isab, “Synthesis, X-ray structure andcytotoxicity evaluation of carbene-based gold(I) complexes ofselenones,” Inorganica Chim. Acta, 2018, 476, 46-53, each incorporatedherein by reference in their entirety.

In view of the need for new anti-cancer agents and pharmacologicaloptions, as disclosed herein the inventors synthesized, characterizedand showed that gold(I)-N-heterocyclic carbene (NHC) complexes exhibitedpotent cytotoxic activity against tumor cells.

BRIEF SUMMARY OF THE INVENTION

The invention involves a series of gold(I)-NHC complexes with thioureaor a substituted thiourea, such as dimethylthiourea; pharmaceuticalcompositions containing these complexes; and methods for inducingcytotoxicity or treating proliferative diseases, disorders or conditionssuch as cancer by administering these gold(I) complexes. The gold(I)complexes of the invention conform to Formula I below:

wherein R1 and R2 are, independently, hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, or C₄₋₁₀ aryl and where the anion is PF₆ ⁻ oranother pharmaceutically acceptable anion.

The foregoing paragraph has been provided by way of generalintroduction, and is not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings below.

FIG. 1. Molecular structure of complex [Au(Ipr)(κSCN₂H₄]PF₆ (1), withlabelling atoms and 50% probability ellipsoids level and withouthydrogen atoms for clarity.

FIG. 2. Molecular structure of complex [Au(Ipr)(κSCN₂C₂H₈]PF₆ (2), withlabelling atoms and 50% probability ellipsoids level.

FIG. 3. Graph of cytotoxic effect of series complexes (0-2)concentrations on cell viability of HCT15 cell line.

FIG. 4. In vitro cytotoxic effect of series complexes (0-2)concentrations on cell viability of HCT15 cell line.

FIG. 5. Graph of cytotoxic effect of series complexes (0-2)concentrations on cell viability of MG-63 cell line.

FIG. 6. In vitro cytotoxic effect of series complexes (0-2)concentrations on cell viability of MG-63 cell line.

FIG. 7. Graph of cytotoxic effect of series complexes (0-2)concentrations on cell viability of HeLa cell line.

FIG. 8. In vitro cytotoxic effect of series complexes (0-2)concentrations on cell viability of HeLa cell line.

FIG. 9A. Cyclic voltammogram of the 0.1 mM complex (1) (A) in 0.1 M PB(pH 6.86).

FIG. 9B. Cyclic voltammogram of the 0.1 mM complex (2) (B) in 0.1 M PB(pH 6.86).

FIGS. 10A-10C. Cyclic voltammograms of the interactions of the complex(1) (FIG. 10A) and complex (2) (FIG. 10B) with 0.5 mM tryptophan in 0.1M PB (pH 6.86) at different concentrations of complexes (1) and 2 (a)blank, absence of complex and tryptophan, (b) 0 μM (c) 10 μM, (d) 20 μM,(e) 40 μM. The response of 0.5 mM tryptophan solution in controlledexperiment (FIG. 10C) by adding solvent blank (b) 0 μL, (c) 15 μL, (d)30 μL, (e) 60 μL.

FIG. 11A: Representative reverse transcription-polymerase chain reaction(RT-PCR) showing β-actin. Caspase 3 and 9 gene expression in HCT-15cells (Human Colon Carcinoma).

FIGS. 11B and 11C. Percent relative intensity measurement of caspase 3(FIG. 11B) and 9 (FIG. 11C) expressions in RT-PCR for each groupexpressed as percentage of β-actin.

FIG. 12A. Representative reverse transcription-polymerase chain reaction(RT-PCR) showing β-actin, Caspase 3 and 9 gene expression in HeLa cells(Human Cervix Cancer).

FIGS. 12B and 12C. Percent relative intensity measurement of caspase 3(FIG. 12B) and 9 (FIG. 12C) expressions in RT-PCR for each groupexpressed as percentage of β-actin.

FIG. 13A. Representative reverse transcription-polymerase chain reaction(RT-PCR) showing β-actin, Caspase 3 and 9 gene expressions in MG-63cells (Human Osteosarcoma).

FIGS. 13B and 13C. Percent relative intensity measurement of caspase 3(FIG. 13B) and 9 (FIG. 13C) expressions in RT-PCR for each groupexpressed as percentage of 3-actin.

FIG. 14 Gold(I) complex of Formula (I).

FIG. 15. Gold(I) complex (1).

FIG. 16. Gold(I) complex (2).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “compound” and “complex” are usedinterchangeably, and are intended to refer to a chemical entity, whetherin a solid, liquid or gaseous phase, and whether in a crude mixture orpurified and isolated.

As used herein, the term “substituted” refers to replacing at least onehydrogen atom of a molecule with a non-hydrogen functional group. Suchnon-hydrogen functional groups can independently include, for example,one or more of the following: alkyl, alkenyl, alkynyl and aryl.

As used herein, the term “alkyl” refers to a fully saturated branched,or unbranched hydrocarbon fragment, preferably for substitution at R1and/or R2 of Formula (I) a C₁-C₆ alkyl. Representative examples of suchalkyl include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl,isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl and the like.

The term “aryl”, as used herein, includes aromatic monocyclic ormulticyclic (e.g., tricyclic, bicyclic), hydrocarbon ring systemscomprising or consisting of hydrogen and carbon atoms, where the ringsystems may be partially saturated. Aryl groups include, but are notlimited to, phenyl, tolyl, xylyl, biphenyl, naphthyl, anthracenyl,phenanthryl and tetralin. This term also includes substituted aryl andheteroaryl groups such as phenol, aryl halides or imidazyl preferably anC₆-C₁₀ aryl is chosen for substitution at R1 and R2 of Formula (I).

The term “halogen”, as used herein, means fluoro, chloro, bromo andiodo.

The term “anion” means a negatively charged ion including, but notlimited to, halides, such as fluoride, chloride, bromide, and iodide,nitrate, sulfate, phosphate, methanesulfonate, ethanesulfonate,p-toluenesulfonate, salicylate, malate, maleate, succinate, tartrate,citrate, acetate, perchlorate, trifluoromethanesulfonate,acetylacetonate, tetrafluoroborate, hexafluorophosphate, andhexafluoroacetylacetonate.

One aspect of the invention relates to a method for treating aproliferative disease, disorder or condition in a subject in needthereof that includes administering to the subject at least one complexcomprising a gold atom coordinated with a thiourea that has thefollowing chemical structure:

wherein R1 and R2 are, independently, hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, or C₆₋₁₀ aryl. These groups include C₁, C₂, C₃,C₄, C₅, Or C₆ alkyl; C₂, C₃, C₄, C₅, Or C₆ alkenyl; and C, C₂, C₃, C₄,C₅, Or C₆ alkynyl, which may be further substituted, for example, withone or more alkyl, alkenyl, alkynyl, halogen, or hydroxyl groups.Advantageously, this method may involve administering a complex such ascomplex (1) wherein R1 and R2 are each hydrogen or a complex such ascomplex (2) wherein R2 and R2 are each methyl.

Any pharmaceutically acceptable anion may for a part of the complex ofFormula 1 including fluoride, chloride, iodide, hexaflurophosphate (“PF₆⁻), or triflate.

Surprisingly, in view of the relatively lower activity of thio-basedcomplexes compared to seleno-based complexes as reported by Molter, etal., Dalt. Trans., 2018, 47, 5055-5064, the inventors found that thegold(I) thiourea complexes of the invention exhibited high degrees ofantiproliferative and cytotoxic activity as shown in the Examples.

While not being bound to any particular theory, the inventors believethat use of thio-based complexes may overcome problems associated withinteraction of other kinds of gold(I) complexes with serum albumin whichmay act as a gold(I) complex scavenger.

As used herein the terms “subject” and “patient” may be usedinterchangeably.

A subject treated with a gold(I) complex of the invention may have aproliferative disease, disorder or condition such as cancer includingsarcoma, carcinoma, lymphoma, or a germ cell tumor. Other types ofcancer which may be treated using this method include cervical cancer,bone cancer, colon cancer, testicular cancer, germ cell cancer, cervicalcancer, bladder cancer, head and neck cancer, esophageal cancer, lungcancer, mesothelioma, a brain tumor or neuroblastoma. Other types ofcancer or proliferative diseases that are currently treated withplatinum-based drugs such as cisplatin may be treated with a gold(I)complex as disclosed herein.

In some embodiments the subject is at least 0, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or >90 yearsof age. A subject may be male or female and may have a newly diagnoseddisease, disorder or condition, be currently under treatment, or sufferfrom a relapsing or chronic disease, disorder or condition. A subjectmay be someone who has lost or is losing responsiveness to anothertreatment, such to a cancer treatment with cisplatin or anotherplatinum-based anticancer agent. A subject may have a comorbid conditionsuch as diabetes, heart disease, hypertension (high blood pressure),hyperlipidemia (high cholesterol) and peripheral vascular disease.

A subject may have experienced one or more side-effects of another kindof treatment, such as side-effects to a cancer treatment with cisplatinor another platinum containing anticancer agent. Side effects includeone or more of the following: bone marrow suppression, neurotoxicity,ototoxicity or hearing problems, nephrotoxicity or kidney problems,electrolyte disturbance, nausea, vomiting, numbness, trouble walking,allergic reactions, electrolyte problems including hypomagnesaemia,hypokalaemia and hypocalcaemia, and/or heart disease.

Those skilled in the art can determine a suitable mode for administeringa gold (I) complex of the invention based on patient status, type ofdisorder, disease or condition, or anatomical location of cellsassociated with the disorder, disease or condition. Advantageously fortreatment of many types of cancer, the gold (I) complex will beadministered intravenously or intraperitoneally. Other modes ofadministration include those that bring the gold complexes into contactwith target cells, such as into contact with proliferating cancer ortumor cells. These modes include oral and parenteral administration.Administration may be targeted to a specific site or anatomicalcompartment containing cancer cells including into a solid cancer orinto the vasculature or other compartments for a non-solid cancer. Othermodes of administration include intravesical, intradermal, transdermal,subcutaneous, intramuscular, intralesional, intracranial, intrapulmonal,intracardial, intrasternal and sublingual modes.

In some embodiments, a gold complex of the invention may be administeredas the only active agent. In other embodiments, more than one type ofgold complex of the invention, such as a cocktail containing bothcomplex (1) and complex (2), may be administered.

In still other embodiments, one or more gold complexes of the inventionmay be administered in conjunction with another agent, such as anotheranti-cancer drug used to treat ovarian cancer, biliary tract cancer,lung cancer (diffuse malignant pleural mesothelioma), gastric cancer,carcinoma of salivary gland origin, breast, colon, lung, prostate,melanoma and pancreatic cancer cell lines, squamous cell carcinoma ofmale genital tract, urothelial bladder cancer, or cervical cancer; orwith at least one drug such as paclitaxel, paclitaxel and 5-FU, UFT(tegafur/uracil), doxorubicin, cyclophosphamide and doxorubicin,gemcitabine, osthold, honeybee venom, anvirzel, or beaciozumab.

Other embodiments of the invention include the gold complexes, per se,as well as compositions, such as those containing one or more goldcomplexes as disclosed herein and encompassed by Formula (I) incombination with a pharmaceutically acceptable excipient or carrier orin combination with another active agent. Typically, a pharmaceuticalcomposition containing a gold(I) complex is sterile, isotonic, andotherwise suitable for administration to a patient.

As used herein, the term “composition” refers to a mixture of the activeingredient with other chemical components, such as pharmaceuticallyacceptable carriers and excipients. The composition may be manufacturedusing any of a variety of processes, including, without limitation,conventional mixing, dissolving, granulating, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilisate,tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosageform suitable for administration.

As used herein, the term “active ingredient” refers to an ingredient inthe composition that is biologically active, for example, the gold(I)complex of Formula (I), a salt thereof, a solvate thereof, a tautomerthereof, and a stereoisomer thereof.

As used herein, the phrase “pharmaceutically acceptable carrier orexcipient” refers to a pharmaceutically acceptable material, compositionor vehicle such as a liquid or solid filler, diluent, binder,manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), or solvent encapsulating material, involvedin carrying or transporting the subject compound from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.

Exemplary materials which can serve as pharmaceutically acceptablecarriers include, but are not limited to: (1) sugars, such as lactose,glucose and sucrose; (2) starches, such as corn starch and potatostarch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powderedtragancanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such ascocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) other non-toxiccompatible substances employed in pharmaceutical formulations andmixtures thereof. Non-limiting examples of specific uses ofpharmaceutically acceptable carriers can be found in, e.g.“Pharmaceutical Dosage Forms and Drug Delivery Systems” (Howard C. Anselet al., eds., Lippincott Williams & Wilkins Publishers, 11^(th) edition,2017; ISBN-13: 978-1496347282); “Remington: The Science and Practice ofPharmacy” (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins,21^(th) edition 2005; 0-7817-4673-6); “Goodman & Gilman's ThePharmacological Basis of Therapeutics” Joel G. Hardman et al., eds.,McGraw-Hill Professional, 13^(th) edition. 2017, 1259584739); and“Handbook of Pharmaceutical Excipients” (Raymond C. Rowe et al., APhAPublications, 5^(th) edition, 2005; 1582120587), each incorporatedherein by reference in their entirety.

In another embodiment, wetting agents, emulsifiers and lubricants, suchas sodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants may also be present inthe compositions described herein. Exemplary pharmaceutically acceptableantioxidants include, but are not limited to: (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

In another embodiment, the pharmaceutically acceptable carrier orexcipient is a binder. As used herein, “binders” refers to materialsthat hold the ingredients in a tablet together. Binders ensure thattablets and granules can be formed with the required mechanicalstrength, and give volume to low active dose tablets. Exemplarypharmaceutically acceptable binders include, but are not limited to: (1)saccharides and their derivatives, such as sucrose, lactose, starches,cellulose or modified cellulose such as microcrystalline cellulose,carboxy methyl cellulose, and cellulose ethers such as hydroxypropylcellulose (HPC), and sugar alcohols such as xylitol, sorbitol ormaltitol; (2) proteins such as gelatin; and (3) synthetic polymersincluding polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG).

Binders can be classified according to their application. Solutionbinders are dissolved in a solvent (i.e. water or alcohol in wetgranulation processes). Exemplary solution binders include, but are notlimited to, gelatin, cellulose, cellulose derivatives,polyvinylpyrrolidone, starch, sucrose and polyethylene glycol. Drybinders are added to the powder blend, either after a wet granulationstep, or as part of a direct powder compression (DC) formula. Exemplarydry binders include, but are not limited to, cellulose, methylcellulose, polyvinylpyrrolidone and polyethylene glycol. In terms of thepresent disclosure, the pharmaceutically acceptable carrier or excipientmay be a solution binder, a dry binder or mixtures thereof.

In some embodiments, the pharmaceutically acceptable carrier and/orexcipient used herein may be an organic solvent, an inorganic salt, asurfactant, and/or a polymer.

Exemplary inorganic salts include, without limitation, calciumcarbonate, calcium phosphate, disodium hydrogen phosphate, potassiumhydrogen phosphate, sodium chloride, zinc oxide, zinc sulfate, andmagnesium trisilicate.

Surfactants that may be present in the compositions of the presentdisclosure include zwitterionic (amphoteric) surfactants, e.g.,phosphatidylcholine, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),anionic surfactants, e.g., sodium lauryl sulfate, sodium octanesulfonate, sodium decane sulfonate, and sodium dodecane sulfonate,non-ionic surfactants, e.g., sorbitan monolaurate, sorbitanmonopalmitate, sorbitan trioleate, polysorbates such as polysorbate 20(Tween 20), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80),cationic surfactants, e.g., decyltrimethyl ammonium bromide,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,tetradecyltrimethyl-ammonium chloride, and dodecylammonium chloride, andcombinations thereof.

Exemplary polymers include, without limitation, polylactides,polyglycolides, polycaprolactones, polyanhydrides, polyamides,polyurethanes, polyesteramides, polyorthoesters, polydioxanones,polyacetals, polyketals, polycarbonates, polyorthocarbonates,polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),poly(amino acids), polyvinylpyrrolidone, polyethylene glycol,polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, orcombinations or mixtures thereof.

The composition may have <0.01, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2,5, 10, 20, 50, 100 or >100 μM of the gold(I) complex of formula (I)relative to the total volume of the composition.

A composition may contain <0.01, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2,5, 10, 20, 50 or >50 wt % of a gold(I) complex of formula (I) based on atotal weight of the composition or any intermediately value within thisrange.

To reduce viability of cancer cells or abnormally proliferating cells, asubject may be treated with a concentration of a gold(I) complexdescribed herein such as complex (1) or (2) ranging from 5, 10, 20, 30,40, 50, 60, 70, 80, 90 or 100 μM or with an amount that contacts thecells in the subject to a concentration of 5, 10, 20, 30, 40, 50, 60,70, 80, 90 or 100 μM of the gold(I) complex.

To reduce proliferation of cancer cells or abnormally proliferatingcells, a subject may be treated with a concentration of a gold(I)complex, such as complex (1) or (2), described herein ranging from 1, 2,5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μM or with an amount thatcontacts the proliferating cells in the subject to a concentration of 1,2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μM of the gold(I)complex. The ranges in the preceding paragraphs include all subrangesand intermediate values. Preferably, a dosage of about 25, 30, 35, 40,45, or 50 μM of a gold(I) complex of the invention is used.

The gold(I) complexes of the invention, such as gold complex (1), may beused to modulate or upregulate the expression of caspaces such asCaspase 3 and 9, and to induce apoptosis in target cells.

Depending on the route of administration e.g. oral, parental, ortopical, the composition may be in the form of solid dosage form such astablets, caplets, capsules, powders, and granules, semi-solid dosageform such as ointments, creams, lotions, gels, pastes, andsuppositories, liquid dosage forms such as solutions, and dispersions,inhalation dosage form such as aerosols, and spray, or transdermaldosage form such as patches.

In other embodiments, the composition having the gold(I) complex ofFormula (I) of, the salt thereof, the solvate thereof, the tautomerthereof may be prepared in a form for immediate release or sustainedrelease.

The term “immediate release” refers to the release of a substantialamount of an active ingredient immediately upon administration.Typically, an immediate release indicates a complete (100%) or less thancomplete (e.g. about 70% or more, about 75% or more, about 80% or more,about 85% or more, about 90% or more, about 95% or more, about 99% ormore, 99.9%, or 99.9%) dissolution of an active ingredient within 1-60minutes, 1-30 minutes, or 1-15 minutes after administration.

The term “sustained release” refers to the release of an activeingredient from a composition and/or formulation over an extended periodof time. In some embodiments, a sustained release indicates adissolution of an active ingredient over a period of time up to 30minutes, 60 minutes, 3 hours, 12 hours, 24 hours upon administration. Inone embodiment, the compositions described herein do not have asustained release.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive ingredients are ordinarily combined with one or more adjuvantsappropriate to the indicated route of administration. If administeredper os, the active ingredients can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also include buffering agents such as sodium citrate, magnesium orcalcium carbonate or bicarbonate. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

The term “parenteral”, as used herein, includes subcutaneous,intravenous, intramuscular, and intrasternal injection, or infusiontechniques. For therapeutic purposes, formulations for parenteraladministration can be in the form of aqueous or non-aqueous isotonicsterile injection solutions or suspensions. These solutions andsuspensions can be prepared from sterile powders or granules having oneor more of the pharmaceutically acceptable carriers or excipientsmentioned for use in the formulations for oral administration. Theactive ingredients can be dissolved in water, polyethylene glycol,propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesameoil, benzyl alcohol, sodium chloride, and/or various buffers. Otheradjuvants and modes of administration are well and widely known in thepharmaceutical art.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a pharmaceutically acceptable diluent or solvent. Amongthe pharmaceutically acceptable diluents and solvents that may beemployed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed, including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid are useful in the preparation ofinjectables. Dimethyl acetamide, surfactants including ionic andnon-ionic detergents, and polyethylene glycols can be used. Mixtures ofsolvents and surfactants such as those discussed above are also useful.

Example S

As shown by the following Examples, water-soluble gold(I) complexes ofthe type [Au(Ipr)(L)]PF₆, where L=Tu (1) or Me₂Tu (2) were synthesizedfrom the parent [(Ipr)AuCl] (0). These complexes (0-2) were fullycharacterized by elemental analysis (EA), FT-IR, ¹H, ¹³C, ⁷⁷Se NMRliquid state and ¹³C solid state. Single crystal X-ray diffractionanalysis shows that both complexes have a linear geometry. The in vitrocytotoxic activity of the complexes and cisplatin was investigated usingan MTT assay against MG-63, HCT15, and HeLa cell lines. The IC₅₀ valuesshowed that the complexes (1) and (2) exhibited a cytotoxicity higherthan cisplatin against all cancer cell lines except complex (2) againstHeLa. The interaction of complexes with amino acids were evaluatedelectrochemically in a phosphate buffer aqueous solution using cyclicvoltammetry. Complex (1) interacted more with L-tryptophan than complex(2). The reduction in peak height and peak current was observed by theinteraction of both complexes with L-tryptophan. The cell deathmechanism was measured by studying the expression levels of Caspase-3and Caspase-9 gene. The treatment of complex (1) with HCT-15 and HeLacells resulted in the induction of apoptosis and a significantupregulation in the expression of both caspase-3 and 9, whereas, nosignificant deviation in the expression was noted in the complex (1)treated MG-63 cells.

Materials, Instruments and Methods.

All chemicals and solvents used in the synthesis were of analyticalgrade and used without further purification. [Au(Ipr)Cl], AgPF₆,thiourea, N,N′-dimethylthiourea, L-tryptophan, sodium phosphate monobasic and disodium phosphate were purchased from Sigma-Aldrich St.Louis, Mo. United States. CH₂Cl₂, methanol and ethanol were purchasedfrom Strem Chemicals, Mass., United States. For experiment and solutionpreparation, double distilled water was used. It was obtained from Labbased Water Still Aquatron A 4000 D unit.

Elemental analyses were performed on Perkin Elmer Series 11(CHNS/O),Analyzer2400. The solid state FTIR spectra of the ligands and theirgold(I) complexes were recorded on a Perkin Elmer FTIR 180spectrophotometer using KBr pellets over the range 4000-400 cm⁻¹ atresolution 4.0 cm⁻¹. Melting point analysis was carried out onBarnstead/Electrothermal (BI) 9100. ¹H and ¹³C NMR spectra recorded on aLAMBDA Jeol 500 spectrophotometer operating at 500.01 and 125.65 MHzrespectively; corresponding to a magnetic field of 11.74 T.Tetramethylsilane(TMS) was used as an internal standard for ¹H and ¹³CNMR measurements. The ¹³C NMR spectra obtained with ¹H broadbanddecoupling, and the spectral conditions were: 32 k data points, 0.967 sacquisition time, and 1.00 s pulse delay and 450 pulse angle. ¹³C (MAS)NMR results recorded on a Bruker 400 MHz spectrometer at ambienttemperature of 25° C. Samples were packed into 4 mm zirconium oxiderotors. Pulse delay of 7.0 s and a contact time of 5.0 ms. The magicangle spinning rates were 4 and 8 kHz. Carbon chemical shifts weremeasured relative to adamantane at 38.56 ppm.

Auto Lab (Netherland) was used as an electrochemical work station forthe cyclic voltammetric experiment. The electrochemical experiment wasperformed using three electrode systems. Platinum as a counter, glassycarbon electrode as a working and Ag/AgCl was used as a referenceelectrode for all electrochemical measurements. The pH and the weightmeasurements of various chemicals were done using Accumet® XL50 pH meterand GR-2000 instruments, respectively.

Synthesis of Gold(I) Complexes.

The complexes were synthesized using a modification of a previouslyreported procedure; see M. Altaf et al. (2014), incorporated herein byreference in its entirety. AgPF₆ (0.127 g, 0.500 mmol) was dissolved in5.0 mL ethanol and added to a solution of1,3-Bis(2,6-di-isopropylphenyl)imidazol-2-ylidenechloridogold(I),[Au(Ipr)(Cl)] (0.311 g, 0.500 mmol) in 5.0 mL CH₂Cl₂. Then stirred for 5min at room temperature and filtered off. Tu (0.038 g, 0.500 mmol) andMe₂Tu (0.052 g, 0.500 mmol) were added to the filtrate, stirred for 1hour and then filtered off. The synthesized complexes were kept in anundisturbed area. After three days, colorless crystals were obtained anda suitable crystal was chosen for X-ray diffraction analysis.

-   -   [Au(Ipr)(κSCN₂H₄)]PF₆ (1). Yield 0.313 g, 75%. C₂₈H₄₀Au F₆N₄SP.        Calcd. C, 41.69, H, 4.99, N, 6.94, S 3.97; found C, 40.04, H,        5.23, N, 6.04, S 3.74. M. P. 243-246° C.    -   [Au(Ipr)(κSCN₂C₂H₈]PF₆ (2). Yield 0.245 g, 61%. C₃₀H₄₄AuF₆N₄SP.        Calcd. C, 43.16, H, 5.31, N, 6.71, S, 3.84; found C, 42.26, H,        5.57, N, 7.20, S 3.66. M.P. 183-186° C.

In some embodiments alternative ingredients, such as AgNO₃ instead ofAgPF₆, solvents such as H₂O or CHCl₃, and a temperature range of 20-27°C. may be used. Some alternative preparation steps include adding Tu(0.038 g, 0.500 mmol) and Me₂Tu (0.052 g, 0.500 mmol) to a solution of1,3-Bis(2,6-di-isopropylphenyl)imidazol-2-ylidenechloridogold(I),[Au(Ipr)(Cl)] (0.311 g, 0.500 mmol) in 5.0 mL CH₂Cl₂ or CHCl₃ andstirred for 2 hours.

Gold (I) complexes of Formula (I) are produced using Tu (thiourea)substituted with hydrogen, C₁_₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, orC₄₋₁₀ aryl. For example, complex (1) and complex (2) as described hereinare respectively produced using thiourea, which has two hydrogensubstituents at R1 and R2, and thiourea substituted with methyl at R1and R2 (dimethylthiourea).

Single Crystal Structure Determination.

Suitable crystals of complexes (1) and (2) were obtained as colorlessplats from dichloromethane/ethanol. The X-ray data were collected at173K (−100° C.) on a STOE IPSD II Image Plate Diffraction Systemconnected with a two-circle goniometer and using a MoKα graphitemonochromator (λ=0.71073 Å); see G. Stoe & Cie, X-Area & X-RED32. Stoe &Cie GmbH, Darmstadt, 2009 Germany, incorporated herein by reference inits entirety. The structure was solved by a SHELXS-2014 program; see G.M. Sheldrick, Acta Cryst., 2008, A64, 112-122, incorporated herein byreference in its entirety. The refinement and further calculations werecarried out with a SHELXL-2014.

The NH H atoms were located in a difference Fourier map and refined witha distance restraint of N—H=0.88(2) Å and H . . . H=1.40(2) Å. TheC-bound H-atoms were included in the calculated positions and treated asriding atoms: C—H=0.95-1.0 Å with U_(iso)(H)=1.5 U_(eq)(C) for methyl Hatoms and =1.2 U_(eq)(C) for other H-atoms. The non-H atoms were refinedanisotropically, using weighted full-matrix least-squares on F². Asemi-empirical absorption correction was applied using the MULscanABSroutine in PLATON; see A. L. Spek, Acta Cryst., 2009, D65, 148-155; andC. F. Macrae, I. J. Bruno, J. A. Chisholm, P. R. Edgington, P. McCabe,E. Pidcock, L. Rodriguez-Monge, R. Taylor, J. Van de Streek, P. A. Wood,J. Appl. Cryst. 2008, 41, 466-470, each incorporated herein by referencein their entirety. The F atoms of the PF₆ ⁻ anion are disordered. Thebest solution was found by distributing the electron density over atotal of 11 positions, which were refined with various fixed occupancyratios to give a total of six F atoms. The symmetry of the crystal dataand structure refinement is demonstrated in Table 6.

MTT Assay for In Vitro Cytotoxicity of Gold(I) Complexes.

Gold(I) complexes (0-2) and cisplatin were tested for their in vitrocytotoxic effects against the MG-63, HCT15 and HeLa cell lines; M.Altaf, M. Monim-ul-Mehboob, A. A. Isab, V. Dhuna, G. Bhatia, K. Dhuna,“The synthesis, spectroscopic characterization and anticancer activityof new mono and binuclear phosphanegold(I) dithiocarbamate complexes,”New J. Chem., 2015, 39, 377-385, incorporated herein by reference in itsentirety.

The cells were seeded at 3×10³ cells/well in 100 μL of DMEM containing10% fetal bovine serum (FBS) in a 96-well tissue culture plate andincubated for 72 h at 37° C., 5% CO₂ and 90% relative humidity in a CO₂incubator. After incubation, 100 μL of (25, 12.5, 6.25 and 3.75 μM)solutions of cisplatin, gold(I) complexes (0-2) prepared in DMEM wereadded to the cells, and then the cultures were incubated for 72 h. Themedium in the wells was casted off and 100 μL of DMEM containing MTT(0.5 mg/ml) was added to the wells, with subsequent incubation in theCO₂ incubator at 37° C. in the dark for 4 h. After incubation,purple-colored formazan produced by the cells appeared as dark crystalsin the bottom of the wells. The culture medium was carefully removedfrom each well to prevent disruption of the monolayer and 100 μL ofdimethyl sulfoxide (DMSO) was added to each well. The solution in thewells was thoroughly mixed to dissolve the formazan crystals whichproduced a purple solution. The absorbance of the 96 well-plates wasmeasured at 570 nm with a LabSystems Multiskan EX ELISA reader against areagent blank. The experimental results were calculated as a micro-molarconcentration of 50% cell growth inhibition (IC₅₀) of each drug. An MTTassay was carried out in three independent experiments, each intriplicate.

Electrochemical Measurements.

The electrochemical measurements were done using a glassy carbonelectrode. For electrochemical analysis, a GCE surface was polished byrubbing on the synthetic cloth containing alumina slurry. The GCEsurface was polished before every electrochemical measurement.Interaction of the complexes was considered with biomolecules usingcyclic voltammetry. The cyclic voltammetry was scanned from 0.0 to 1.2or −0.4 to 1.2 V.

Semi-Quantitative Reverse Transcription-Polymerase Chain Reaction.

The underlying molecular mechanism for the anti-cancer activity ofdifferent gold complexes was determined. The gene expression of caspasefamily proteins i.e. caspase 3 (CASP3) and caspase 9 (CASP9) wasexamined by constructing a first transcript (cDNA) from their respectivemRNA by reverse transcription. Further, the cDNA was amplified andelectrophoresed on 2% agarose gel. β-actin (ACTB) was used as aninternal control. The primer pairs for the desired genes werecustom-synthesized from Bioserve Biotechnologies (Hyderabad, India) asshown in Table 1.

TABLE 1 Primers sequence used for semi-quantitative RT-PCR Sr. PCR no.mRNA Primers Sequence (5′→3′) Product 1 CASP9 ForwardATGATCGAGGACATCCAGCG 266 bp (SEQ ID NO: 1) Reverse CTGGGTGTTTCCGGTCTGAG(SEQ ID NO: 2) 2 CASP3 Forward CTCGGTCTGGTACAGATGTCG 263 bp(SEQ ID NO: 3) Reverse ACTTCTACAACGATCCCCTCTG (SEQ ID NO: 4) 3 ACTBForward TCACCCACACTGTGCCCATCTA 295 bp CGA (SEQ ID NO: 5 ReverseCAGCGGAACCGCTCATTGCCAA TGG (SEQ ID NO: 6)

IR Spectroscopy.

The IR frequency bands at 842 and 985 cm⁻¹ were assigned to the C═Sstretching vibration of 1 and 2, which is significantly shifted to alower frequency with respect to their position in free Tu and Me₂Tu(1040 and 1080 cm⁻¹). The shift indicates a decrease in the double bondcharacter of C═S due to the mesomeric effect of the neighboring nitrogenatoms. The N—H stretching shows a significant shift to a higherfrequency (3471, 3369; 3377 Vs 3405, 3274; 3267 cm⁻¹). See D. N.Sathyanarayana, “Vibrational Spectroscopy: Theory and Applications,” NewAge Intl. Publishers, 1st Edition, 2004; K. Aktiviti, A. Kompleks, C.Li, “Synthesis, characterisation and antibacterial studies of Cu(II)complexes thiourea,” malaysain J. Anal. Sci., 2012, 16(1), 56-61; J. T.J. Prakash, L. R. Nirmala, “Synthesis, spectral and thermal propertiesof bis thiourea zinc Acetate (BTZA) single crystals,” intl. J. Comput.Appl., 2010, 6(7) 7-11; and U. Anthoni, G. Borch, P. Klaboe, P. H.Nielsen, “Tentative assignments of fundamental vibrations of thio- andselenoamides. VIII. 1,2-Dimethyl-3-pyrazolidineselone, a cyclicselenohydrizide. selenation of the thioamide group in theory andpractice,” Acta Chem. Scand. A, 1982, 36, 69-77, each incorporatedherein by reference in their entirety. The C═S group forms aconsiderably stronger bond than that of C═Se. See P. J. Hendra, Z. Jović“The laser Raman and infra-red spectra of some thiourea and selenoureacomplexes of platinum(II) and palladium(II),” Spectrochim. Acta, 1968,24, 1713-1720; H. Rostkowska, L. Lapinski, A. Khvorostov, M. J. Nowak,“Proton transfer processes in selenourea: UV-induced selenone→selenolphotoreaction and ground state selenol→selenone proton tunneling,” chem.phys., 2004, 298, 223-232; and N. N. Golovnev, A. A. Leshok, G. V.Novikova, A. I. Petrov “Stability of Bismuth(III), Indium(III),Lead(II), and Cadmium(II) monocomplexes with selenourea and thiourea,”Russ. J. Inorg. Chem., 2010, 55, 130-132, each incorporated herein byreference in their entirety. Weak signals at 2960 & 3073 cm⁻¹ for[Au(IPr)Cl] and 2869,2963; 2873, 2931 & 3162, 3166 cm⁻¹ for 1 and 2 dueto C—H stretching vibrations of IPr were also observed. The IR data forfree ligands and complexes is shown in Table 2.

TABLE 2 Mid FT-IR frequencies (cm⁻¹) of free ligand and Au(I) complexes(0-2) Stretch Stretch Ligand/Complex NH C═S [Au(Ipr)Cl] — 3073 1109 29601366 1258 1462 — Tu 3405, 3274 — — — — 1616 1468 1080 1 3471, 3369 31661059 2963, 2869 1466 1630 1466 842 Me₂Tu 3267 — — — 1444 1525 — 1040 23377 3162 1133 2931, 2873 1371 1571 1462 985

NMR Spectroscopy.

The ¹H and ¹³C NMR chemical shifts of ligands and their complexes aregiven in Tables 2 and 3. The ¹H chemical shifts associated with the IPrpart of land 2 were observed in the same region as the [Au(IPr)Cl]reported in the literature [41, 40], while the N—H resonance of Tu andMe₂Tu in 1 and 2 (7.28 and 8.16 ppm) shifted downfield by about 1.5 ppmcompared to their values in the free ligands.

TABLE 3 ¹H NMR chemical shifts (ppm) for free ligands and gold(I)complexes (0-2) in CDCl₃. Ligand/complex H-3 H-4 H-5 H-6 H-7 H-8 H-9 N—H[Au(Ipr)Cl](0) 7.39 d 7.55 t 2.46 m 1.33 d 1.21 d 7.98 s — — 1 7.36 d7.62 t 2.49 m 1.30 d 1.24 d 6.91 s — 7.28 2 7.35 d 7.52 t 2.50 m 1.26 d1.21 d 7.84 s 2.80 s 8.16 Tu — — — — — — — 6.81 Me₂Tu — — — — — — 2.81 s6.62

A ¹³C NMR chemical shift of the Au═C resonance in the [Au(Ipr)Cl] wasobserved at 175.3 ppm and it was shifted downfield by ≈6.0 ppm in 1 and2 at 180.3 and 181.1 ppm, respectively. The downfield shift was relatedto a back donation from the d-orbital of the Au atom to π* C═S and thetransferred electron density of N→C resulting in a partial doublecharacter in the C—N bond which confirms that the coordination is donethrough binding to the sulfur atom; see A. S. Vinogradov, A. B.Preobrajenski, A. Knop-Gericke, S. L. Molodtsov, S. A. Krasnikov, S. V.Nekipelov, R. Szargan, M. Hävecker, R. Sch{umlaut over (l)}ogl,“Observation of back-donation in 3d metal cyanide complexes through N Kabsorption spectra,” J. Electron Spectrosc. Relat. Phenomena., 2001,116, 813-818, incorporated herein by reference in its entirety.

The other resonances of the IPr ligand remained almost unchanged.However, the C═S resonance in 1 and 2 shifted up-field by 6.0 and 10 ppmwith respect to its position in the free ligands as reported in theliterature; see A. A. Isab, S. Ahmad, “Complexation of(trimethylphosphine)gold(T) with selenones,” Trans. Met. Chem., 2006,31, 500-503, incorporated herein by reference in its entirety.

TABLE 4 13C NMR chemical shifts (ppm) for free ligands and gold(I)complexes (0-2) in CDCl₃. Ligand/ complex C1 C2 C3 C4 C5 C6 C7 C8 Au═CC═S C11 [Au(Ipr)Cl](0) 145.5 133.9 123.0 130.7 28.8 24.5 24.0 124.2175.3 — — 1 145.3 133.1 123.7 129.8 28.1 23.8 23.3 — 180.7 177.0 — 2145.9 133.3 124.0 131.1 28.8 24.5 23.9 124.4 181.1 176.4 32.3 Tu — — — —— — — — — 185.5 — Me₂Tu — — — — — — — — — 182.3 30.8

Single Crystal X-Ray Structure of Complexes (1 and 2).

The molecular structure of complexes [Au(IPr)(κSCN₂H₄]PF₆ (1)[Au(IPr)(κSCN₂C₂H₈]PF₆ (2) are shown in FIGS. 1 and 2. Selected bondlengths and angles are illustrated in Table 5. The geometry of thecoordination around gold ion is close to linearity, with (C—Au—S) angleof 177.12(12) and 176.40(7)° respectively. The Au—C and C—S bond lengthsare 2.2977(7); 1.724(3); 1.995 (4)Å; 1.706 (6), respectively. The bondlengths and bond angles are similar to those reported for analogouscompounds. See O. E. Piro, R. C. Piatti, A. E. Bolzan, R. C. Salvarezza,“X-ray diffraction study of copper(I) thiourea complexes formed insulfate-containing acid solutions,” Acta Crystallogr. B, 2000, 56,993-7; M. Fettouhi, M. I. M. Wazeer, A. A. Isab, “Crystal structure ofdibromo-bis(1,3-imidazolidine-2-thione-S)zinc(II),” z. Krist. NCS, 2006,221, 221-222; M. Fettouhi, A. Isab, M. Wazeer, “Crystal structure of bis(3,4,5,6-tetrahydropyrimidine-2(1H)-thione-S)gold(I) chloride,[Au(C₄H₈N₂S)₂]Cl,” z. Krist. NCS, 2004, 219, 2004; and F. Caddeo, V.Fernandez-moreira, M. Arca, A. Laguna, V. Lippolis, M. C. Gimeno, “Goldthione complexes,” inorganics, 2014, 2, 424-432, each incorporatedherein by reference in their entirety. There is no Au—Au bond in bothcomplexes.

TABLE 5 Selected bond lengths and bond angles for complexes (1) and 2Bond Length (Å) Bond Angles (°) Complex (1) Au1—S1 2.3024 (13) C1—Au1—S1177.12 (12) Au1—C1 1.995 (4) C28—S1—Au1 103.6 (2) S1—C28 1.706 (6)N3—C28—N4 118.6 (6) N3—C28 1.320 (9) S1—C28—N3 123.0 (4) N4—C28 1.321(7) S1—C28—N4 118.40 (4) Complex (2) Au1—S1 2.2977 (7) C4—Au1—S1 176.40(7) Au1—C4 2.007 (2) Au1—S1—C1 105.79 (10) S1—C1 1.724 (3) N1—C1—N2119.20 (3) N1—C1 1.326 (4) S1—C1—N1 122.40 (2) N2—C1 1.322 (4) S1—C1—N2118.40 (2)

TABLE 6 Summary of crystal data and details of the structure refinementfor complex (1) and 2 Complex 1 2 Empirical formula C₂₈H₄₀AuF₆N₄PS C₃₀H₄₄ AuF₆N₄PS Formula weight 806.63 834.69 Crystal symmetry MonoclinicMonoclinic Space group P 21/n P 21/c Crystal color Colorless Crystalsize/mm 0.65 × 0.39 × 0.20 0.40 × 0.32 × 0.20 Wavelength/Å   0.71073  0.71073 Temperature/K 110  173 a (Å)   9.2495(8)   8.6566 (2) b (Å) 15.474 (14)  23.5651 (9) c (Å)  23.370 (2)  17.3653 (5) α (°)  90.0 100.394 (5) β (°)  96.419 (2)  98.966 (2) γ (°)  90.0  95.727 (5) Cellvolume (Å³) 3324.0 (5) 3499.12 (19) D_(x) (g m⁻³)   1.612   1.686 μ(mm⁻¹)   4.594   4.37 Radiation type Mo Kα Mo Kα Z   4   4Diffractometer Bruker Smart STOE IPDS 2 Apex area detector AbsorptionMulti-scan Multi-scan correction SADABS; (MULABS in Sheldrick, 1996PLATON, (2009) Radiation source Plane graphite R[F² < 2σ(F²)], 0.051,0.160, 1.07 0.020, 0.047, 1.01 wR(F²), S Δρ_(max) (e Å⁻³)   2.09   0.90Δρ_(min) (e Å⁻³)  −3.31  −0.58 H-atom treatment: Treated by a mixture ofindependent and constrained refinement

In Vitro Cytotoxic Activities of Gold(I) Complexes.

Gold(I) complexes (0-2) were tested for in vitro cytotoxicity againstHeLa (human cervical cancer), MG-63 (human osteosarcoma (human bonecancer)), and HCT15 (human colon cancer) cancer cell lines using an MTTassay and compared with cisplatin (a standard anticancer drug). Therespective IC₅₀ values (μM) of the gold(I) complexes (0-2) and cisplatinagainst cancer cell lines are shown in Table 7.

The results showed a significant reduction in cell viability with asubsequent increase in the concentration of gold complexes and cisplatin(p<0.005). However, the gold complexes in comparison to cisplatin werefound to be highly cytotoxic at concentrations 100 and 50 μM. Tocalculate the IC50 value for the different complexes, the testedconcentrations were reduced to 25 μM and to 3.12 μM (FIGS. 3-6). TheIC₅₀ values were obtained from the curve of the concentration ofcisplatin, and the complexes (0-2) against the percentage of cellviability.

Complex (1) has both better and lowered IC₅₀ values, and was thereforeselected for the analysis of the underlying molecular mechanism ofcytotoxicity. The data shows that complex (1) is the most active atinhibiting all cell cancer proliferation. Its antiproliferative activityagainst HCT15 (6.64 μM), MG-63 (1.76 μM), and HeLa (9.20 μM) was 2 to17-fold better than cisplatin (30.2, 33.58, 20.55 μM) respectively.

The inhibition of cell proliferation and the higher cytotoxic effect ofthe complexes suggest that the S containing ligand is more labile andthat it prevents the complexes from reacting with the thiol group ofproteins and enzymes, such as albumin, which enhance the cytotoxicactivity of the complexes.

TABLE 7 IC₅₀ values (μM) of cisplatin and gold(I) complex (1) and 2against HCT15, A549 and MCF7 cancer cell lines Complex HCT15 MG-63 HeLaCisplatin 31.94 ± 0.55 33.37 ± 1.2  21.59 ± 0.73 0 119.82 ± 10.6  61.59± 3.65 171.56 ± 11.98 1  6.61 ± 0.18  1.76 ± 0.16  9.17 ± 0.18 2 23.89 ±1.21 14.7 ± 1   42.66 ± 5.94

Interactions of the Complexes with Amino Acids.

Complexes (1) or (2) were evaluated electrochemically to observe theirinteractions with biomolecules. The complexes exhibited good solubilityin water. The electrochemical behavior of the complexes was investigatedusing cyclic voltammetry. For this purpose, 0.1 mM complexes of 1 and 2were scanned from −0.4 to 1.2 (FIG. 9). The complexes were found to beelectrochemically stable and no peaks were observed in the scannedwindow. The electrochemical inactivity of the complexes restricted theexamination of its interaction with electrochemically inactivebiomolecules or proteins. To investigate the interaction of thecomplexes with biomolecules, the electroactive one was selected. Fewamino acids, such as L-tyrosine, and the tryptophan, are electroactiveamino acids; see M. Fettouhi (2006); and N. Baig, A-N Kawde, “A novel,fast and cost effective graphene-modified graphite pencil electrode fortrace quantification of L-tyrosine”, Anal. Methods., 2015, 7, 9535-9541,each incorporated herein by reference in their entirety. The interactionof complex (1) and (2) was explored with tryptophan.

The interaction of the synthesized complexes with tryptophan wasevaluated by spiking various concentrations of complexes (1) and (2)into 0.5 mM of tryptophan. The tryptophan peak appeared in a 0.1 Mphosphate buffer at 0.758 V in the absence of the complexes. In FIG.10A, the interaction of complex (1) could be seen with tryptophan. Asignificant decrease in the peak current was observed. A similarbehavior was observed with the complex (2) (FIG. 10B). The spiking ofvarious concentrations of complex (2) shows an obvious change in thepeak shift and peak current. The peak of tryptophan was shifted from0.758 to 0.803 V by spiking 40 μM of complexes (1) and (2). However,complex (1) demonstrated more effect on the peak shift and the peakcurrent of the tryptophan. The peak of tryptophan was shifted to 0.850V. The possible interaction is due to the presence of the amino group inthe complexes which may interact through forming some sort ofintermolecular forces. To eliminate the possible effect of the solvent,the equivalent volume of the solvent blank was added into the 0.5 mMtryptophan. In a controlled experiment, no effect on the peak shift andthe current was observed as the spiked solvent blank maximum addedvolume is very small (FIG. 10C). It was observed that the change in thepeak shift and the peak current is due to the interactions of the spikedcomplexes (1) and (2). The electrochemical study revealed that bothcomplexes have a good capability to interact with amino acids andproteins.

Semi-Quantitative Reverse Transcription-Polymerase Chain Reaction.

The toxic attribute of complex (1) was further determined by studyingthe expression levels of the Caspase-3 and the Caspase-9 gene, and withthe apoptotic markers at the mRNA level (FIGS. 11-13). The levels ofboth the caspases were measured as a percent relative intensityexpression to β-actin of the respective groups. In comparison to thecontrol group, the treatment with complex (1) of the HCT-15 and HeLacells resulted in the induction of apoptosis and a significantupregulation in the expression of both caspase-3 and 9 was demonstratedat the mRNA level (p<0.005). However, no significant deviation in theexpression of caspase-3 and 9 was noted in complex (1) and the controltreated MG-63 cells (FIG. 5). The expression of the caspases in both thegroups at the mRNA level is represented in the form of a histogram. Theresults represent the mode of the toxic effect of complex (1) in theHCT-15 and HeLa cells. The present results are in line with earlierstudies representing the toxic attribute of gold complexes by theup-regulated expression of caspase-3 and caspase-9; see Y. Wang, Q. Y.He, R. W. Sun, C. M. Che, “Gold(II) porphyrin 1a induced apoptosis bymitochondrial death pathways related to reactive oxygen species,” CancerRes., 2005, 56(24), 11553-64; O. Rackham, S. J. Nichols, P. J. Leedman,S. J. Berners-Price, A. Filipovska “Gold(I) phosphine complexselectively induces apoptosis in breast cancer cells: implications foranticancer therapeutics targeted to mitochondria. Biochemicalpharmacology,” Biochem. Pharmacol., 2007, 74(7), 992-1002; I. Ott, X.Qian, Y. Xu, D. H. Vlecken, I. J. Marques, D. Kubutat, J. Will, W. S.Sheldrick, P. Jesse, A. Prokop, C. P. Bagowski, “A gold(I) phosphinecomplex containing a naphthalimide ligand functions as a TrxR inhibitingantiproliferative agent and angiogenesis inhibitor,” J. Med. Chem.,2009, 52(3), 763-70; and X. Cheng, P. Holenya, S. Can, H. Alborzinia, R.Rubbiani, I. Ott, S. Wölfl. “A TrxR inhibiting gold(I) NHC complexinduces apoptosis through ASK1-p38-MAPK signaling in pancreatic cancercells.,” Mol. Cancer, 2014, 13(1), 221, each incorporated herein byreference in their entirety. However, complex (1) may have induced celldeath by some other mechanisms in spite of the caspase-3 and 9 in theMG-63 cells.

As shown herein the inventors have synthesized, characterized, anddemonstrated the in vitro cytotoxicity of complexes (1) and (2). The ¹³CNMR solid state was used as a complementary technique to explore thestability of the complexes in a liquid and a solid state. Single crystalX-ray diffraction revealed that both complexes have a distorted lineargeometry. Furthermore, complex (1) was found to be more potent as ananticancer agent than cisplatin against all human cancer cell lines.Both complexes showed great stability in an aqueous solution. Complex(1) interacted more with L-tryptophan than complex (2). A reduction inpeak height and a peak current shift in potential were observed by theinteraction of both complexes with L-tryptophan. The mode of toxiceffect of complex (1) in the HCT-15 and HeLa cells induced the cellsdeath by the up-regulated expression of caspase-3 and caspase-9. Complex(1) may have induced cell death by another or an additional mechanism inspite of the caspase-3 and 9 in the MG-63 cells.

Abbreviations

Me₂Tu=N,N′-dimethylthiourea, Tu=thiourea,IPr=1,3-Bis(2,6-di-isopropylphenyl)imidazol-2-ylidene, HeLa=cervicalcancer cell line, HCT15=colon adenocarcinoma, MG-63=bone cancer cellline, NHC=N-hetero-cyclic carbene, DMEM=Dulbecco's Modified Eagle'sMedium.

Terminology

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

The headings (such as “Background” and “Summary”) and sub-headings usedherein are intended only for general organization of topics within thepresent invention, and are not intended to limit the disclosure of thepresent invention or any aspect thereof. In particular, subject matterdisclosed in the “Background” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

Links are disabled by deletion of http: or by insertion of a space orunderlined space before www. In some instances, the text available viathe link on the “last accessed” date may be incorporated by reference.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the materials, compositions, devices,and methods of this technology. Similarly, the terms “can” and “may” andtheir variants are intended to be non-limiting, such that recitationthat an embodiment can or may comprise certain elements or features doesnot exclude other embodiments of the present invention that do notcontain those elements or features.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper”, “in front of” or “behind” and the like, may be used herein forease of description to describe one element or feature's relationship toanother element(s) or feature(s) as illustrated in the figures. It willbe understood that the spatially relative terms are intended toencompass different orientations of the device in use or operation inaddition to the orientation depicted in the figures. For example, if adevice in the 8s is inverted, elements described as “under” or “beneath”other elements or features would then be oriented “over” the otherelements or features. Thus, the exemplary term “under” can encompassboth an orientation of over and under. The device may be otherwiseoriented (rotated 90 degrees or at other orientations) and the spatiallyrelative descriptors used herein interpreted accordingly. Similarly, theterms “upwardly”, “downwardly”, “vertical”, “horizontal” and the likeare used herein for the purpose of explanation only unless specificallyindicated otherwise.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

The invention claimed is:
 1. A method for treating a cancer that isresistant to or unresponsive to cisplatin in a subject in need thereof,comprising: administering to the subject at least one complex comprisinga gold atom coordinated with a thiourea that has the following chemicalstructure:

wherein R1 and R2 are, independently, hydrogen, C_(1,6) alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, or C₆₋₁₀ aryl, and wherein the anion is fluoride,chloride, iodide, hexaflurophosphate (“PF₆ ⁻”), or triflate, wherein thecomplex is intravenously administered to the subject in the form of anaqueous solution.
 2. The method of claim 1, wherein R1 and R2 are eachhydrogen.
 3. The method of claim 1, wherein R1 and R2 are each methyl.4. The method of claim 1, wherein the anion is fluoride, chloride,iodide, or triflate.
 5. The method of claim 1, wherein the anion ishexafluorophosphate (“PF₆−”).
 6. The method of claim 1, wherein thecancer is cervical cancer.
 7. The method of claim 1, wherein the canceris bone cancer.
 8. The method of claim 1, wherein the cancer is coloncancer.
 9. The method of claim 1, wherein the cancer is at least one ofcervical cancer, testicular cancer, germ cell cancer, bladder cancer,head and neck cancer, esophageal cancer, lung cancer, mesothelioma,and/or a brain tumor or neuroblastoma.
 10. The method of claim 1,further comprising administering at least one other anticancer drug usedto treat ovarian cancer, biliary tract cancer, lung cancer (diffusemalignant pleural mesothelioma), gastric cancer, carcinoma of salivarygland origin, breast, colon, lung, prostate, melanoma and pancreaticcancer cell lines, squamous cell carcinoma of male genital tract,urothelial bladder cancer, or cervical cancer.
 11. The method of claim1, further comprising administering at least one of paclitaxel,paclitaxel and 5-FU, UFT (tegafur/uracil), doxorubicin, cyclophosphamideand doxorubicin, gemcitabine, osthold, honeybee venom, anvirzel, andbeaciozumab.